MOLECULAR BIOLOGY LAB B474 LAB 1 RESTRICTION …

MOLECULAR BIOLOGY LAB B474 LAB 1 RESTRICTION ENZYME DIGESTION

The purpose of this lab is to introduce two techniques commonly used in molecular biology: restriction enzyme digestion of DNA, and characterization of DNA by

agarose gel electrophoresis. These techniques will be routinely used in other experiments throughout the rest of the semester.

Restriction endonucleases are enzymes which recognize and cleave double

stranded DNA at specific sequences, and are named for the cellular strain from which they are isolated. The natural function of the enzymes is to destroy foreign DNA, while

the cell's own DNA is protected from cleavage because it has been specifically modified by cellular methylases. Typically, the recognition sites consist of 4-6 (or more) bases

arranged in a palindromic sequence. For example, the enzyme Eco R1 recognizes the site shown, cutting at the arrows, and generating sticky ends:

5---GAATTC---3 3---CTTAAG---5

EcoR1--> 5---G3

&

3---CTTAA5

5 AATTC---3

3

G---5

In this lab we will digest the plasmid pBluscript with the enzyme Eco R1. Bluescript has been completely sequenced, and contains about 3000 bp, with the single

Eco R1 site found in the poly cloning site). The digested samples are subjected to agarose gel electrophoresis, and the DNA fragments are visualized by ethidium bromide staining and exposure to UV light (~310 nm). By varying the concentration of agarose,

one can separate varying ranges of DNA fragment sizes.

We will also digest lambda virus DNA with the enzyme Hind III. This digest will produce a series of fragments which will appear as a ladder on the gel. This particular digest is often used as a standard to size other DNA fragments. The fragment sizes

can be found in most molecular biology catalogues, along with maps of other plasmids and lambda DNA. You can also look in the catalogues to determine where Hind III cuts.

EXPERIMENTAL PROCEDURES (All work is done individually, but 2 people must share each gel.)

MATERIALS pBluescript (100 ng/ul) Eco R1 lambda DNA (250 ng/ul) Hind III Digest Buffer (10 x stock) provided by manufacturer, note formula sterile H2O

microfuge tubes microcentrifuge

Water bath 37?C pipettes (P20 and P200) with sterile tips ice and buckets

heat temp block TAE buffer: 50 x Stock

agarose ethidium bromide (10mg/ml)

loading dye (6x stock): electrophoresis equipment

transilluminator camera with polaroid 667 film or KODAK digital recorder

Special notes that will be important to know:

-

Tubes MUST be labeled by writing your initials, date and sample name

or number on the side and covering with clear tape.

-

Restriction enzymes are very sensitive to temperature changes. Keep them on

ice and avoid warming and contamination by handling the middle of the tube, not at the

bottom or top.

-

While 50% glycerol is present to stabilize the enzyme and to prevent freezing,

the final glycerol concentration in the digest mixture must be less than 5%. Therefore,

the total enzyme volume must be less than 10% of the total digest volume.

-

One unit of enzyme is defined as the amount of enzyme required to digest 1 ug

of lambda DNA to completion in one hour at optimum conditions. (Conditions vary for

each enzyme, and it is important to note the specific requirements of each before

setting up reactions.)

-

Ethidium bromide is a mutagen, so wear gloves whenever handling solutions or

the stained gel.

-

UV light causes severe eye and skin damage. You must wear UV shields or

goggles when viewing the transilluminator.

PROTOCOL

Follow the protocol as written, but use the flow chart to help you visualize how to get everything done in an organized fashion.

1) Set up the digest reaction as shown below (write it in your notebook this way). Also record the ng of DNA present in each tube. Use a fresh tip when adding a different solution to the tube.

tube

______ 1 pBlue(__ng) control

ul BUFFER* ul H2O

H

B

___________ _____

--

2

13

ul DNA pBlue lambda

_______________

5

--

ul Enzyme Eco R1 Hind III

______________

--

--

2 pBl/Eco (__ng) --

2

12

5

--

1

--

3 Lambda (__ng)

control

2

--

16

--

2

--

--

4 Lambda/Hind

2

--

7

--

10

--

2

* buffer names vary by company. We might have different sources, so be careful and ask about the buffers.

2) Spin each sample for a few seconds in the microfuge, and incubate in a water bath at 37?C 60 min.

3) Set up agarose gel as shown. Make 100 ml of a 1% gel in 1x TAE in a 250 ml

erlenmeyer. Melt the agarose in the microwave slowly, so it does not boil over. Let the agarose cool to 60?C, pour into your plate, and insert the comb. The comb should not

touch the bottom of the plate or the wells will not form properly. Pour a gel 4-6 mm deep.

4) When the digest is complete, add 4 ul of 6x loading dye to each tube and heat in the temp. block a few min at 65?C. (The 6x dye should now be diluted to a final concentration of 1x or more. The dye acts as a marker and increases the density of the sample so it can settle into the well). Chill on ice and spin again.

5) Each pair will be given a control lambda Hind III MW ladder as a standard for DNA fragment sizes. Add 1 ul dye per 5 ul of ladder, and heat and spin as shown. (Load this

sample in the center lane of the gel.)

6) When the gel is firm remove the comb (lift comb from one side) and set the gel and tray into the electrophoresis chamber. Fill the reservoir with 1x TAE buffer until the gel is submerged. Flush the wells with a few ul of buffer.

7) Carefully load as much sample as you can into the wells, noting the location of each sample on a strip of tape. (This tape will later be used to tape the gel photo into your notebook and you don't have to rewrite everything!) Put the top on the apparatus, and hook up the apparatus to the power supply, so that the DNA is moving toward the + charge. Run the gel at ~80V. Try not to electrocute yourself.

8) When the blue marker dye is 2/3 of the way down the gel stop migration by turning down the voltage meter and shutting off the power. Stain the gel in ~200 ml H20 containing 50 ul ethidium bromide. Visualize on the transilluminator, record on polaroid film. examine the fragment sizes, and compare to known values.

FLOW CHART -------------------

SET UP DIGEST

DIGEST AT 37?C

MAKE AGAROSE

COOL TO 65?C, POUR GEL

SPIN SAMPLES

PREPARE FOR GEL LOADING

LOAD SAMPLES INTO GEL

RUN GEL AT 80 Volts 60 MIN.

STAIN 5 MIN

VISUALIZE AND PHOTOGRAPH GEL

Determine size of fragments

MOLECULAR BIOLOGY PREP SHEET

WEEK 1 RESTRICTION ENZYME DIGEST DATE NEEDED: AS SOON AS POSSIBLE, *ENZYMES CAN NOT BE ALIQUOTED

UNTIL JUST PRIOR TO LAB.

ITEM

AMOUNT NEED ( per 20)

TOTAL(20)

Bluescript ul

25 ul per pair

Eco R1

6 ul per pair

lambda DNA 250 ng/ul

23 ul per pair

Hind III

6 ul per pair

Digest Buffer H and B

10 ul per pair

(or appropriate)

control lambda ladder

1 tube per pair

sterile H2O

5 bottles per lab

TAE buffer: 50xStock

1 liter

agarose

1 bottle

ethidium bromide (10mg/ml)

1 bottle

loading dye (6x stock):

1 rack of 10 tubes, filled

microfuge tubes

several beakers full

microcentrifuge

all available

Water bath 37?C

1 big one

floating racks

4 per lab

pipettes (P20 and P200)

10 each type

sterile tips

10 racks

ice and buckets

5

heat temp block

all

weigh boats and spatulas

electrophoresis equipment

1 per pair

(power supplies and units)

transilluminator

1

camera

1

with polaroid 667 film Or KODAK

UV shields

gloves, all sizes

scissors

scotch tape

marking pens

lab tape

250ul (25ug) 60ul 230ul (75ug)

10 sets all 1 box each

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